Human dendritic cells adenovirally-engineered to express three defined tumor antigens promote broad adaptive and innate immunity

Abstract

Dendritic cell (DC) immunotherapy has shown a promising ability to promote anti-tumor immunity in vitro and in vivo. Many trials have tested single epitopes and single antigens to activate single T cell specificities, and often CD8(+) T cells only. We previously found that determinant spreading and breadth of antitumor immunity correlates with improved clinical response. Therefore, to promote activation and expansion of polyclonal, multiple antigen-specific CD8(+) T cells, as well as provide cognate help from antigen-specific CD4(+) T cells, we have created an adenovirus encoding three full length melanoma tumor antigens (tyrosinase, MART-1 and MAGE-A6, "AdVTMM"). We previously showed that adenovirus (AdV)-mediated antigen engineering of human DC is superior to peptide pulsing for T cell activation, and has positive biological effects on the DC, allowing for efficient activation of not only antigen-specific CD8(+) and CD4(+) T cells, but also NK cells. Here we describe the cloning and testing of "AdVTMM2," an E1/E3-deleted AdV encoding the three melanoma antigens. This novel three-antigen virus expresses mRNA and protein for all antigens, and AdVTMM-transduced DC activate both CD8(+) and CD4(+) T cells which recognize melanoma tumor cells more efficiently than single antigen AdV. Addition of physiological levels of interferon-α (IFNα) further amplifies melanoma antigen-specific T cell activation. NK cells are also activated, and show cytotoxic activity. Vaccination with multi-antigen engineered DC may provide for superior adaptive and innate immunity and ultimately, improved antitumor responses.

Figure 1. T cell activation by AdVTMM1 transduced DC. (A) AdVTMM1/DC T cell specificity. Healthy donor T cells were activated with AdVTMM1-transduced DC, expanding CD8⁺ and CD4⁺ T cells specific for each of the three encoded antigens, as tested by HLA-restricted peptides, in the IFNγ and IL-2 ELISPOT assays. (B) AdVTMM1/DC T cell recognition of antigen-expressing melanoma cells. Seven of nine individual cultures stimulated with AdVTMM/DC recognized Mel526 by IFNγ ELISPOT at a superior frequency than AdV-single antigen/DC, from three different healthy donors. Five of the seven positive cultures are shown. (C) AdVTMM1/DC^+/− IFNα. Five of five AdVTMM1/DC cultures testing addition of IFNα showed superior melanoma recognition by ELISPOT, compared with AdVTMM alone. Paired cultures expanded at the same time from the same donor are shown together (B and C).

Figure 2. The AdVTMM2 cloned virus produces mRNA and protein for all three antigens. mRNA expression in AdVTMM2 was tested in 293 cells which were transduced with AdVTMM2 or left untransduced (negative control). Antigen expressing Mel526 cells served as a positive control for tyrosinase and MART-1. A549 served as a positive control for MAGE-A6. RNA was isolated and underwent RT-PCR to detect mRNA for each antigen. The same groups were tested for protein by protein gel blot, with B-actin as a loading control. Two tests with the 5B57 antibody for MAGE-A6 are shown.

Figure 3. AdVTMM2 transduced DC activate CD8⁺ and CD4⁺ T cells specific to all three antigens. Antigen specificity was tested with T cells from four different healthy donors which were activated with AdVTMM2-transduced DC. The cultures expanded CD8⁺ and CD4⁺ T cells specific for each of the three encoded antigens, as tested by HLA-A2 and DR4-restricted peptides (Table S1), in IFNγ and IL-2 ELISPOTS (A and B) in five different experiments. In the case of HLA-DR4-negative donors, total CD4⁺ T cell responses to each antigen were determined by testing recognition of autologous DC transduced with single antigen AdV and control AdVLacZ (C).

Figure 4. Maturation of DC prior to AdVTMM2 transduction enhances immunogenicity. AdVTMM2/DC stimulated cultures were tested with and without DC pretreatment with IFNγ + LPS. After three weekly stimulations, the total frequency of antigen-specific lymphocytes (without T cell subset purification) producing IFNγ was tested in two donors. Reactivity to autologous DC loaded with Mel526 lysate was also compared.

Figure 5. AdVTMM/DC activate NK cells. Phenotype and cytotoxicity of NK cells from two different donors was tested by flow cytometry. (A) CD56^hi/CD16^- and CD56⁺/CD16⁺ NK cells from unstimulated PBMC and from cultures stimulated with AdVMAGE A-6/DC, AdVTMM1/DC and AdVTMM1/DC + IFNα were stained for surface CD25 and CD69 and intracellularly stained for granzyme B content. AdVTMM2/DC cultures also contained > 95% CD69⁺ NK cells (not shown). (B) The ability to upregulate 7-AAD on the LAK target Daudi (labeled with CTO) was compared between unstimulated NK cells and NK cells isolated from AdVTMM2/DC cultures. Three different ratios of NK cells to Daudi targets were tested (10:1, 50:1 and 100:1).